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Abstract

Background

The liver presents a remarkable capacity for regeneration after hepatectomy but the
exact mechanisms and mediators involved are not yet fully clarified. Erythropoietin
(EPO) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have been shown
to promote liver regeneration after major hepatectomy.

Aim of this experimental study is to compare the impact of exogenous administration
of EPO, GM-CSF, as well as their combination on the promotion of liver regeneration
after major hepatectomy.

Methods

Wistar rats were submitted to 70% major hepatectomy. The animals were assigned to
4 experimental groups: a control group (n = 21) that received normal saline, an EPO
group (n = 21), that received EPO 500 IU/kg, a GM-CSF group (n = 21) that received
20 mcg/kg of GM-CSF and a EPO+GMCSF group (n = 21) which received a combination of
the above. Seven animals of each group were killed on the 1st, 3rd and 7th postoperative
day and their remnant liver was removed to evaluate liver regeneration by immunochemistry
for PCNA and Ki 67.

Results

Our data suggest that EPO and GM-CSF increases liver regeneration following major
hepatectomy when administered perioperatively. EPO has a more significant effect than
GM-CSF (p < 0.01). When administering both, the effect of EPO seems to fade as EPO
and GM-CSF treated rats have decreased regeneration compared to EPO administration
alone (p < 0.01).

Conclusion

EPO, GM-CSF and their combination enhance liver regeneration after hepatectomy in
rats when administered perioperatively. However their combination has a weaker effect
on liver regeneration compared to EPO alone. Further investigation is needed to assess
the exact mechanisms that mediate this finding.

Introduction

Liver has the unique capacity to regain its original and optimal mass after partial
hepatectomy [1]. However the risk of immediate postoperative hepatic failure, especially if the procedure
is performed in patients with a diseased liver, still represents a barrier to the
extent of hepatectomy that can be attempted. The identification of factors that enhance
liver regeneration and their clinical implication could reduce the morbidity and mortality
associated with liver surgery.

However, liver regeneration is a complex phenomenon and the implicated mechanisms
are not yet fully understood and clarified. It is well known that mature hepatocytes
can replicate [1], representing the main mechanism of hepatocyte production during regeneration, as
well as non-parenchymal cells that are located in the liver [2]. Bone marrow cells may also play a role in the generation of hepatocytes after liver
injury, while it is known that many cytokines like IL-6 and TNFa and growth factors
like TGFa, EGF and HGF are implicated in different stages of the regenerative process
[3,4].

In studies that have been performed in the past, erythropoietin (EPO) has been shown
to be produced by the regenerating liver after partial hepatectomy in rats [5] and erythropoietic foci have been found 24-72 hours after subtotal hepatectomy in
rats [6]. EPO has been shown to have a positive effect on liver regeneration after hepatectomy
in many studies [7,8]. In addition, EPO has been found to have a positive effect on liver regeneration
after ischemia and reperfusion injury [9-11].

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a cytokine that, besides
the proliferation and differentiation of haemopoietic precursor cells, has additional
effects on the functional properties of mature cells involved in inflammation and
immunity [12]. It also enhances the functions of mature macrophages that are induced to secrete
various cytokines including IL-6 and TNF-a, substances known to participate in liver
regeneration [13]. GM-CSF has been used in the past, in order to stimulate liver regeneration following
hepatectomy [14].

The combined administration of EPO and GM-CSF could possibly have a cumulative effect
on liver regeneration. As this is an appealing intervention in order to enhance liver
regeneration after hepatectomy, there are reports suggesting an antagonistic relationship
between the two factors [15,16].

Aim of the present study is to compare the effect of the administration of EPO and
GM-CSF alone or in combination on the acceleration of liver regeneration in rats after
major hepatectomy.

Materials and methods

Adult male Wistar rats weighing 200-250 gr each were obtained from the Hellenic Pasteur
Institute (Athens, Greece) after the approval of the study protocol by Aretaieion
Hospital Research Committee and the authority of the Athens prefecture for experimental
protocols. They had free access to food and water and were kept in an air-conditioned
room at 21°C with a 12-hr/12-hr light-dark cycle. The animals were fasted for 12 hr
before the procedure and the same care continued in the postoperative period. Care
and handling was in accordance with the National and European guidelines laboratory
animal care.

Eighty four wistar rats were submitted to 70% major hepatectomy. The animals were
assigned to 4 experimental groups: a control group (n = 21) that received normal saline,
an EPO group (n = 21), that received EPO 500 IU/kg, a GM-CSF group (n = 21) that received
20 mcg/kg of GM-CSF and an EPO+GMCSF group (n = 21) which received a combination of
the above. EPO, GM-CSF or normal saline were administered subcutaneously every day
at 7 am for 8 days before the operation and for 2 days postoperatively.

For the induction of anesthesia 40 mg/kg ketamine (Ketalar 10 mg/ml) along with 1
mg/kg of atropine (atropine sulfate 1 mg/ml) were injected intramuscularly. Moreover,
in a different side 5 mg/kg of Midazolam (Dormicum 15 mg/3 ml) diluted to 0.4 ml of
normal saline 0,9% were also injected in order to maintain long lasting anesthesia
of the animals undergoing liver resection. The surgery consisted of 70% partial hepatectomy
according to the methods described by Higgins and Anderson [17]. The operations were performed between 9 am and noon. The resected liver was sampled
for immunohistochemical study in order to evaluate if the factors that were administered
for 8 days before hepatectomy had any effect on hepatocytes and to serve as self-control.
Seven animals of each group were killed under anaesthesia by exsanguination on postoperative
days 1, 3 and 7. Immediately after exsanguination the liver was removed for the study
immunohistochemical study of regeneration.

Hepatic regeneration was evaluated by immunohistochemistry for Proliferating Cell
Nuclear Antigen (PCNA) and Ki-67 [18]. Immunostaining of liver specimens was performed by using an anti-PCNA monoclonal
antibody (PC-10, Dakopatts, Glostrup, Denmark). The three-step immunoperoxidase method
using the Streptavidin-Biotin complex (Dakopatts) was performed, according to a procedure
described previously [19]. Ki 67 was stained using a mouse anti-rat Ki-67 antibody (Dako, Denmark). Tissue
sections were inspected at high power (x400 magnification) by two independent pathologists
in a blind-coded manner. Positive nuclei were counted in 5-10 randomly chosen fields
that approximate 1000 hepatocytes per section. The intensity of the staining was evaluated
as negative, medium and high, the latter two being accepted as positive. Data were
expressed as the percentage of cells that were positively stained.

The weight of the animals the day of surgery and the day of euthanasia was also recorded.

Statistical Analysis

Data are expressed as mean ± SD. Differences between groups were analyzed by one-way
analysis of variance (ANOVA), or if the data were not normally distributed by a Kruskal-Wallis
ANOVA on ranks. Differences between time points of the same group were analyzed with
univariate ANOVA. Bonferroni correction was used for post hoc multiple group comparisons.
The level of statistical significance was defined as p < 0.05.

Postoperative day 1

On postoperative day 1 all rats had increased Ki 67 and PCNA expression (p < 0.05).

Postoperative day 3

On postoperative day 3 all rats had increased Ki 67 (p < 0.05). PCNA was increased
in the EPO and GM-CSF+ EPO groups, while there was no increase in the GM-CSF group.

Postoperative day 7

One week after hepatectomy, hepatocytes showed increased expression of PCNA in all
groups (p < 0.01), while Ki-67 was increased only in the EPO treatment group (p <
0.01).

In all postoperative days, the combination of EPO and GM-CSF failed to increase PCNA
and Ki 67 staining to the extent that EPO alone did (p < 0.01). Both markers did not
have any difference between the groups treated with GM-CSF and the combination of
EPO and GM-CSF. In addition both Ki 67 and PCNA expression were significantly increased
in EPO compared to GM-CSF treated animals in all post-operative days (p < 0.01). The
results are summarized in Figures 1 and 2.

Figure 1.Ki-67 expression. Percentage of Ki-67 expression for each experimental group. Data are expressed as
mean ± standard deviation. * p < 0.05 compared to baseline of the same timepoint.
† p < 0.01 compared to baseline of the same timepoint. ‡ p < 0.01 compared to EPO
group of the same timepoint. ◊ p < 0.01 compared to EPO group of the same timepoint.

Figure 2.PCNA expression. Percentage of PCNA expression for each experimental group. Data are expressed as
mean ± standard deviation. * p < 0.05 compared to baseline of the same timepoint.
† p < 0.01 compared to baseline of the same timepoint. ‡ p < 0.01 compared to EPO
group of the same timepoint. ◊ p < 0.01 compared to EPO group of the same timepoint.

The percentage of postoperative total body weight variation did not differ significantly
between groups as shown in Figure 3.

Figure 3.Body weight. Postoperative variation of total body weight. Data are expressed as mean ± standard
deviation. * p < 0.05 compared to postoperative day 3 of the same group.

During the experiments 11 rats died, either due to hemorrhage or by immediate postoperative
complications. These rats were excluded from the study and replaced by other animals.

Discussion

Liver presents a remarkable capacity for proliferation after a partial hepatectomy
and can precisely regulate its growth and mass to adjust its size. The exact mechanisms
of stimulation and regulation of hepatic regeneration remain unclear. It is well known
that various cytokines and growth factors and perhaps cell populations, other than
hepatocytes are involved. Many different substances have been reported to stimulate
liver cell growth in vivo and in vitro, including a number of known hormones, serum
factors and some small nutrient molecules [1,2,4].

The discovery that EPO and its receptor play a significant biological role in tissues
outside of the hematopoietic system has provoked significant experimental interest
and fueled the exploration of additional actions of the hormone [20,21]. It is a member of the class I cytokines family and is considered a pleiotropic hormone.
The EPO-specific receptor has been recognized in different cells, such as endothelial
cells, epicardium, placenta, pancreatic islets, renal cells and defined areas of the
brain [22]. Previous studies suggested that erythropoietin (EPO) was produced in rats by the
regenerating liver [6] following partial hepatectomy and erythropoietic foci have been recognized 24-72
hours after subtotal hepatectomy in rats [6]. Kupffer cells seem to be the site of erythropoietin production after hepatectomy
[5].

Angiogenesis seems to be a fundamental requirement for liver regeneration and its
regulation. The modulation of endothelial cell proliferation or apoptosis has been
shown to affect liver regeneration after partial hepatectomy in mice [23]. During liver regeneration the expression and activity of proapoptotic pathways is
inhibited and after massive liver resection the activation of apoptosis is a major
cause for failure of regeneration [24]. Recently EPO has been found to inhibit apoptosis after injury in various organs,
like the brain [25], kidney [26] and the myocardium [27].

GM-CSF is a haematopoietic growth factor that apart from stimulating the proliferation
and differentiation of myeloid bone marrow progenitor cells, also enhances the function
of mature macrophages that are induced to secrete various cytokines including IL-6
and TNF-a [13]. It has been found to be a very potent immunostimulating agent by priming macrophages
to produce cytokines, like TNF-a and IL-6 in blood of healthy humans as well as in
blood of immunosupressed patients with sepsis and after cardiopulmonary bypass [28]. Within minutes after PH, Kupffer cells release cytokines, specifically TNF-a and
IL-6 that are substantial for hepatocytes priming and preparation for replication
[3]. Eroglu et al have already shown that GM-CSF promotes liver regeneration after hepatectomy
in normal and cirrhotic livers [14].

The above mentioned experimental evidences prompted us to compare the effects of the
administration of rhEPO, GM-CSF and their combination on liver regeneration following
major hepatectomy. Although the effect of EPO in this setting has already been reported,
there are few data on the effect of GM-CSF. In addition there are no data on the effect
of their combination on liver regeneration. These two factors are thought to be mitogens
and their combination should have a cumulative regenerating effect on the liver. However,
Fatouros et al have reported that their combined administration seems to attenuate
the beneficial role of EPO on intestinal anastomosis healing, which is similarly a
mitotic process [16]. The major end-point of this study was to investigate if their combination has a
synergistic or antagonistic effect on liver regeneration after major hepatectomy.

In our study we chose to evaluate the expression of two proliferation markers -PCNA
and Ki 67-, as these have been shown to peak at different timepoints of the cell cycle,
and their expression could vary depending on the stage of cellular duplication. They
are sensitive markers of hepatocyte proliferation, which correlate well with the extent
of regeneration [18]. In addition, they are have already been widely used for the study of liver regeneration
and in particular for the study of the effects of EPO on liver regeneration.

Our study demonstrates that EPO administration had a positive effect on liver regeneration
process after 70% hepatectomy by augmenting nuclear activity. This effect is noted
even before any "triggering" for regeneration took place, as rats pre-treated with
rhEPO showed increased expression of both Ki 67 and PCNA before hepatectomy was performed.
This is in accordance with the literature, as Bockhorn et al have also demonstrated
similar results. They reported that EPO preconditioning for three days can raise significantly
the Ki-67 proliferation index and liver-to-body weight ratio of the normal liver [7]. In addition proliferation markers were increased after hepatectomy until 3 days
on rats treated with rhEPO, similarly to our results [8]. Although the increase in our study is substantial, it is the result of a prolonged
EPO pretreatment period (8 days). In addition, our results represent to total amount
of hepatocytes stained, whether the staining was moderate or intense. As Ki 67 antigen
is expressed during the whole cell cycle, it is uncertain whether the moderately stained
cells are in the process of mitosis, or the antigen is still expressed in the cell
after mitosis.

The dose of EPO administered in our study was 500 IU/kg and was administered subcutaneously.
A wide variety of doses have been used by other authors [7-9,29,30]. We used the doses used by Fatouros et al in a study trying to compare the combined
effect of EPO and GM-CSF on colonic anastomoses healing [15,16]. Generally they are considered low doses in this experimental setting. However we
did not want to use higher doses as they have been shown to inhibit liver regeneration
[29].

In our study, pre-operative GM-CSF administration resulted in increased hepatocyte
proliferation before hepatectomy, as well as at postoperative days 1 and 7. Preoperatively
only PCNA was over-expressed, and not Ki 67. This can be explained by the fact that
these two markers of cellular proliferation do not correspond to the same cell cycle
phase, as PCNA concentration seems to peak at the at the S phase of the cell cycle,
while Ki 67 peaks later, during mitosis, in the M phase [18]. Eroglu et al showed increased hepatocyte proliferation 2 days after hepatectomy
in rats were GM-CSF was administered. This effect however faded at the 7th postoperative day. However in their study GM-CSF was administered immediately after
hepatectomy, while in our study we pretreated animals for 8 days before hepatectomy
and 2 days after [14]. The dose of GM-CSF administered was 20 mcg/kg as used by other authors [16].

On the other hand pretreatment with the combination of EPO and GM-CSF resulted in
a weaker proliferative response compared with animals that were treated with EPO alone.
Since EPO alone increased nuclear activity, it would seem logical that the combination
group would have the same results. The fact that this group showed less nuclear activity
than the EPO group, suggests perhaps a competitive action between the two growth factors.
This is in accordance with the findings of other studies, where although EPO administration
increased the tensile strength of colonic anastomoses postoperatively in rats, the
combined administration of EPO and GM-CSF failed to show the same results [15,16]. Many possible mechanisms have been proposed in the literature. GM-CSF may play an
antagonistic role on the EPO receptor as these hemopoietins have a high homology [31,32]. A competition between EPO and GM-CSF has been reported in cells of the marrow [33]. In addition, it has been shown that GM-CSF can modulate EPO effects in certain leukemic
cell line models of hematopoiesis, modulating events at the transcriptional and signal
transduction level, or decreasing mRNA levels of EPO receptor [34]. Finally concentrations of hemopoietins have been found to play a key role in the
final effect on cellular response [35].

Conclusions

In conclusion our data suggest that EPO and GM-CSF, when administered perioperatively
in hepatectomy are able to accelerate liver regeneration. This can be added to the
apparent beneficial effect of EPO in reducing blood transfusions that are associated
with increased morbidity and might be of particular clinical interest in situations
where hepatectomy is expected to result in significant liver failure and increased
mortality. Future research can focus on the effect of these factors after hepatectomy
when hepatic parenchymal disease coexists, as well as on the role of autologous transfusion
inducing endogenous EPO production. Finally, the mechanisms involved in the inhibition
of EPO by GM-CSF are the focus of our current research.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

VI, LE and SV conceived of the study, and participated in its design and coordination
and helped to draft the manuscript. NC, TA and DN conducted the experiments. TT and
FG participated in the design of the study and performed the statistical analysis,
FM and KA performed the immunohistochemistry assays. All authors read and approved
the final manuscript.